American Journal of Pathology, Vol. 138, No. 4, April 1991 Copyright C American Association of Pathologists
Expression of Neural Cell Adhesion Molecule in Normal and Neoplastic Human Neuroendocrine Tissues Long Jin,* John J. Hemperly,t and Ricardo V. Lloyd* From the Department of Pathology, * University of Michigan, Ann Arbor, Michigan; and Becton Dickinson and Company, t Research Triangle Park, North Carolina
The neural cell adhesion molecule (N-CAM) is a group of cell surface glycoproteins involved in direct cell-cell adhesion. N-CAM expression in normal and neoplastic tissues was examined with specific antibodies and oligonucleotide probes by immunohistochemistry and in situ hybridization. Most neuroendocrine cells and tumors with secretory granules expressed N-CAM protein and mRNA Parathyroid adenomas (4) were somewhat unusua* because N-CAM mRNA, but not proteing was detected in some of these benign neoplasms. Most non-neuroendocrine cells and tumors did not express N-CAM, although uterine smooth muscle and an adrenal cortical carcinoma were both positive. Western blots disclosed proteins of 180, 140, and 120 kd in normal adult brain; whereas two pheochromocytomas, a null cell adenoma and a gastrinoma had proteins of approximately 180 and 140 kd. These results indicate that N-CAM protein and mRIVA are widely expressed in neuroendocrine cells and neoplasms. N-CAM oligonucleotide probes as well as antibodies against N-CAM can be used as broad-spectrum neuroendocrine markers. In addition; these molecular probes can be used to examine the role of N-CAM in the development and regulation of neuroendocrine tissues. (Am J Pathol 1991, 138:961-969)
The neural cell adhesion molecule (N-CAM) is a group of cell surface glycoproteins involved in direct cel-cell adhesion.1" Cloning and sequencing of the cDNA for N-CAM indicates that N-CAM is a member of the immunoglobulin gene superfamily.4 Various forms of N-CAM have been found in vertebrate species such as chickens, mice, rats, and humans. In adult brain, N-CAM consists of three major polypeptides with Mr 180, 140, and 120 kd,
which are generated by alterative RNA splicing from a single gene.5'6 The sequence of the coding regions of the N-CAM gene has been well conserved during
evolution.1' 3 N-CAM originally was described as a D2 protein and brain-specific antigen-2 (BSP-2).7 It was later demonstrated that N-CAM, D2, and BSP-2 were immunochemically identical.9 The expression of N-CAM is regulated in a developmental stage and tissue-type-specific
manner.1012 In early embryos, the cells of all three primitive layers express N-CAM, whereas in adults N-CAM expression is more restricted. The expression of N-CAM in adult tissues was previously considered to be mainly restricted to the nervous system. Recent studies have shown, however, that N-CAM was also expressed in some non-neural tissues,1-16 including some endocrine cells.17'18 A few reports have described the presence of N-CAM in selected normal and pathologic human tissues.1'23 Moolenaar et al19'20 reported that an antiN-CAM monoclonal antibody 735 and small cell lung carcinoma (SCLC) cluster 1 monoclonal antibodies recognized identical glycoproteins in SCLC. The Leu-7-antigen on lung tumor cells, which is recognized by HNK-1 monoclonal antibody, is a carbohydrate epitope found on N-CAM, as well as on a number of other cell adhesion molecules.24'25 Recently polyclonal antisera against human N-CAM were prepared by one of us (John J. Hemperly). Using affinity-purified antisera, we have analyzed the distribution of N-CAM by immunohistochemistry (IHC) and immunoblot methods in neuroendocrine tissues. In addition, the DNA sequence of human N-CAM has been determined by the characterization of cDNA clones encoding N-CAM from a human neuroblastoma cDNA library.26 Various oligonucleotide probes to N-CAM were developed, and the distribution of N-CAM mRNA was analyzed by in situ hybridization (ISH). In this study, we have analyzed the distribution of N-CAM expression in Supported in part by NIH grant CA 37238. Accepted for publication December 19, 1990. Address correspondence to R. Lloyd, MD, PhD, Department of Pathology, University of Michigan Medical Center, 1500 E. Medical Center Dr., Room 2G332/Box 0054, Ann Arbor, Ml 48109-0054.
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normal and neoplastic human tissues at both protein and mRNA levels. The results showed a wide distribution of N-CAM expression in human neuroendocrine tissues.
Materials and Methods Tissues Fifty-five normal and neoplastic human tissues from mostly surgical specimens and a few autopsy specimens from the Department of Pathology were used in these studies (Table 1). Tissues were frozen in liquid nitrogen and kept at - 700C for up to 2 years. For IHC and ISH studies, the tissues were embedded in optimal cutting temperature (OCT) medium and approximately 6- to 8- -thick sections were cut in a cryostat at - 200C. Sections used for IHC were mounted on poly-l-lysine-coated slides and fixed in acetone at room temperature for 10 minutes, and then kept in phosphate-buffered saline (PBS) until immunostaining was carried out. The sections used for ISH were mounted on 3-aminopropylmethoxysilane-coated slides, followed by fixation in 4% paraformaldehyde, pH 7.2, for 20 minutes. The slides were then washed in 2x SSC (1 x SSC = 0.15 mol/l [molar] NaCI and 0.015 mol/l Na citrate), dehydrated in 95% and 100% ethanol, and stored at - 700C for up to 1 month.
Immunohistochemistry The frozen sections were immunostained as previously reported using the ABC method27 (Vector Kits, Burlingame, CA). An immunoaffinity-purified rabbit polyclonal anti-human N-CAM antibody was used at 1/50 1/100 on all slides. The immunoaffinity-purified anti-N-CAM antibodies were prepared as follows. Human brain N-CAM was purified by immunoaffinity chromatography of a detergent extract on a column of monoclonal antibody antiLeu19 (Becton Dickinson) coupled to Sepharose. AntiLeul 9 reacts with N-CAM as well as a subset of cytotoxic T cells in the peripheral blood. Bound material was eluted with 0.1 mol/l diethanolamine (pH 11), 0.5% NP-40, and immediately neutralized with sodium phosphate. Purified N-CAM was coupled to Reacti-Gel 6X (Pierce Chemical Co.) as recommended by the manufacturer. Briefly, the relatively dilute N-CAM eluate from the anti-Leu19Sepharose was dialyzed against 0.1 molA sodium borate (pH 8.5) and mixed with the beads. After absorbing overnight, the beads were washed and stored. To purify the antibodies, an antisera prepared by immunizing rabbits with N-CAM purified as above was rocked with about 1 ml of N-CAM-Reacti-Gel for 1 hour in the cold. The column was washed with PBS. The antibodies were eluted with 3 ml of pH 11 buffer without detergent and neutralized with 2 mol/l sodium phosphate. The protein concen-
Table 1. N-CAM Immunobistochemical and In Situ Hybridization Analys* of Nornmal and Neoplastic Human Tissues In situ Tissues Diagnosis Immunohistochemistry hybridization
Pituitary Adrenal
Cerebral cortex Cerebellum Thyroid
Parathyroid Pancreas
Normal Null cell Adenoma Prolactin adenoma Normal Pheochromocytoma Cortical Carcinoma Neuroblastoma Paraganglion/paraganglioma Normal Normal Medullary carcinoma Adenocarcinoma Adenoma Normal Insulinoma/gastrinoma
Stomach Lung
Carcinoid Carcinoid Small cell carcinoma
Skin Breast Ovaries Uterus
Merkel cell carcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenomyosis (myometrium) Adenocarcinoma Normal Normal Squamous cell carcinoma
Colon Liver Skin
* Number of positive cases/number of total cases examined. t Represent focal positive staining in less than 25% of the tumor cells.
3/3* 4/4 1/1 3/3 4/4 1/1 2/2 3/3 1/1 2/2 3/3 0/2 0/4 2/2 2/2 1/1 1/1 1/1 1/1 3/4t 0/3 0/1 1/1 0/2 0/1 0/1 0/1
3/3* 4/4 1/1 3/3 4/4 1/1 2/2 3/3 1/1 2/2 3/3 0/2 2/4' 1/2 2/2 0/1 1/1 1/1 1/1 3/4t 0/3 0/1 1/1 0/2 0/1 0/1 0/1
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tration of the antibody was about 80 ,ug/ml. Normal rabbit serum diluted 1/100 was substituted for primary antibody as a negative control.
Probes and Labeling An oligonucleotide probe that recognizes messenger RNA (mRNA) coding for N-CAM was prepared on the basis of human N-CAM cDNA sequence. This 30-base oligonucleotide probe was complementary to human NCAM, encoding for amino acids 808 through 817 of the cytoplasmic domain: 5' d(GGC TTC GTT TCT GTC TCC TGG CAC TCT GGC)-3'.26 This sequence was present on N-CAM 180 and 140. The probe was prepared using an Applied Biosystems (Foster City, CA) 381 A DNA synthesizer and purified using an OPC cartridge (Applied Biosystems). The oligonucleotide probe was labeled by the 3'-end labeling method as previously described,28 using 100 ng of oligonucleotide incubated with 45 ,uCi of [35S] dCTP or [35S] dATP (1300 1500 ci/mmol), 2 mmol/I (millimolar) CaCI2, 0.5 mmol/I dithiothreitol, and 20 units of terminal deoxynucleotidyl transferase in 100 mmol/l potassium cacodylate (pH 7.2) for 45 minutes at 370C. The labeled probe was purified by gel electrophoresis on 12% polyacrylamide gels as previously described.28.
and 100% ethanol with 0.3 mol/l ammonium acetate, and air dried. Autoradiography was carried out by dipping in Kodak NTB2 emulsion diluted 1:1 with distilled water containing 0.3 moVI ammonium acetate. The slides were allowed to air dry for 1 hour and then placed in a 40C dessicated chamber for 1 week. They were subsequently developed for 3 minutes in Kodak D-19 developer, washed in water for 1 minute and fixed for 5 minutes in Kodak Fixer. After washing in water for 1 hour, they were stained with hematoxylin and eosin (H&E), and coverslipped. The results of ISH were analyzed under the microscope by using dark-field and bright-field illumination. The control for ISH consisted of 1) prior digestion of frozen sections with 100 ,ug/ml of RNAse A (Sigma) at 370C for 45 minutes before ISH; 2) omission of specific probes in hybridization buffer for ISH; 3) Using a sense DNA oligonucleotide N-CAM probe. Quantitation of ISH data was done by taking photographs at a final magnification of x 1000. Grains per cell were enumerated using a total of 100 to 150 cells. Results were expressed as number of silver grains per cell.
-
In Situ Hybridization Slides were incubated in 100% and 95% ethanol for 5 minutes each and washed in water at room temperature. The slides were then treated with 0.25% acetic anhydride in triethanolamine (vol/vol) for 10 minutes followed by incubation in 2x SSC for 30 minutes at 700C, then washed in 2 x SSC at room temperature for 5 minutes before covering the slides with 40 ,ul of hybridization buffer (2x SSC containing Denhardt's solution [0.02% Ficoll/0.02% bovine serum albumin/0.02% polyvinylpryrrolidine], 10% dextran sulfate, 125 ,ug/ml of yeast tRNA, 100 RIl/ml of sonicated salmon sperm DNA, and 50% formamide in 20 mmol/I sodium phosphate [pH 7.2]) for 1 hour at room temperature. The labeled probes were then diluted in hybridization buffer containing 20 mmol/I dithiothreitol and 1 x 1 o6 cpm of probe/slide. They were covered with coverslips treated with Sigmacote and incubated overnight at 440C in moist chambers. After 18 hours, the coverslips were gently removed in 2x SSC and the slides were washed at room temperature in 2x SSC for 2 hours, then in 1 x SSC for 1 hour and 0.5x SSC for 30 minutes. A subsequent wash in 0.5x SSC for 30 minutes at 440C was followed by a 30-minute wash at room temperature in 0.5x SSC. The slides were then dehydrated in 95%
Immunoblot Proteins were extracted with 1% NP40 or 1% sodium dodecyl sulfate, and separated on a polyacrylamide gel, transferred to nitrocellulose paper, and immunostained with a polyclonal N-CAM antibody."9 30 Proteins were not treated with neuraminidase before Western blotting.
Results
Immunohistochemistry Most normal and neoplastic neuroendocrine tissues showed diffuse or focal immunoreactivity with the polyclonal N-CAM antiserum, including pituitary adenomas, pheochromocytomas, neuroblastomas, medullary thyroid carcinomas, and other endocrine tumors from lung, pancreas, gastrointestinal tract, and skin (Table 1) (Figures 1 A-F). Immunoreactivity appeared to be associated with the cytoplasmic membranes, in some cases with better morphologic preservation of tissues (Figures 1 C, D). The normal adrenal medullae and pheochromocytomas were strongly labeled, but adrenal cortical cells were negative (Figure 1A, B). One adrenal cortical carcinoma examined, however, was strongly positive. Normal pituitary (some secretory cells of adenohypophysis and the neurohypophysis) (Figure 1 C), brain (the neuron, astrocytes, and fiber bundles), and cerebellum (the Purkinje cells and granular layer) from autopsy were also intensely stained. All cells of the pancreatic islets of Langerhans
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Figure 1. A: (top left) Immunoristocbemknal staining of an acetone-fixed frozen section of adrenal gland showing a positive reaction for N-CAM protein in the medulla while the cortex is negative (x250). B: (top right) Pheochromocytoma localizing N-CAM tumor cells (X400). C: (middle left) Localization of N-CAM in the adenoypopbysis of normal pituitary tissues. Many of the cells stained positively (X350). D: (middle right) N-CAM immunoreactivty in a medullary thyroid carcinoma (x350). E: (bottom left) Merkel cell carcinoma sbowing positive staining for N-CAM in mosttumor cells (x300). F: (bottom right) Parathyroid adenomas did not sbow positive immunoreactivity with the
N-CAM antibody (x300).
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were stained. Medullary thyroid carcinomas (Figure 1 D) and a Merkel cell carcinoma (Figure 1 E) were positive. All four cases of the parathyroid adenomas were completely negative after immunostaining (Figure 1 F). In contrast, most nonendocrine tissues, such as liver, skin, adult muscle, colon, and ovarian carcinomas did not react with the N-CAM antiserum. Smooth muscle from a case of uterine adenomyosis stained positively, whereas the blood vessel smooth muscle in the same case was negative. Several cases of breast carcinomas had focal immunostaining of the epithelial cells. Extracellular staining was also seen on some elements of the basement membrane, collagen fibrils, and some lymphocytes, as well as nerve fibers.
Immunoblot N-CAM determinants were analyzed in different tissues by immunoblot using the polyclonal N-CAM antibody (Figure 2). Pituitary null cell adenomas, gastrinomas, and two pheochromocytomas were positive with two major bands of approximately 180 kd and 140 kd. Adult brain, used as a positive control, had three positive bands of 180, 140, and 120 kd. Liver tissue, a parathyroid adenoma, and a medullary thyroid carcinoma were negative by immunoblot studies. The extra bands present in the Western blots in lanes 1 and 6 and in the two pheochro-
mocytomas (lanes 3 and 4) were detected consistently with the polyclonal antiserum and with a battery of monoclonal antibodies to N-CAM. These bands probably represent variation in molecular sizes due to differential glycosylation.
In Situ Hybridization Analysis of the distribution of N-CAM mRNA by ISH with the N-CAM oligonucleotide probe is summarized in Table 1 (Figures 3 to 5). The results of ISH demonstrated that most endocrine tissues expressed N-CAM mRNA, which correlated with the IHC analysis. Silver grains were present over the nucleus and the cytoplasm (Figures 3 through 5). Pheochromocytomas, neuroblastomas, and pituitary null cell adenomas had stronger hybridization signals than other endocrine tissues, as judged by the number of silver grains per cell (Figure 6). The normal adrenals and pituitaries had weaker hybridization signals compared with the tumors that originated from these tissues. Positive hybridization signals were found focally in two of four parathyroid adenomas. No ISH signals were detected in most non-neuroendocrine tissues. Hybridization with the sense N-CAM probe did not produce a positive hybridization signal (Figure 3B). Omission of the oligonucleotide probe or pretreatment with RNAse before ISH abolished the hybridization signal.
Discussion
211 -
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Figure 2. Immunoblot showing the expression of various N-CAM species in endocrine tumors and adult brain tissues. Lane 1 is a pituitary null cell adenoma: lane 2 is a gastrinoma; lanes 3 and 4 are pheochromocytomas; lane 5 shows normal liver and lane 6 shows normal adult brain. Three hundred micrograms ofprotein was used in lanes 1 to 5, and 100 jig ofprotein was used in lane 6 The molecular weight standards are shown on the left.
The present studies demonstrate that N-CAM polypeptides and mRNA are present in many neuroendocrine cells and tumors. Early studies suggested that N-CAM expression was restricted to cells of neural crest lineage, such as neurons, glial cells, chromaffin cells of the adrenal medullary, and some tumor cell lines derived from these.2'710 Because neuroendocrine cells have many features in common with neural cells such as the production of various hormones and amines, we extended the study of N-CAM distribution to many neuroendocrine tissues. The adrenal medulla, paraganglia, neurohypophysis, and C cells of the thyroid are neuroendocrine tissues with a neural crest origin, whereas the neuroendocrine cells from pancreas, gastrointestinal tract, and lung are not of neural crest origin.31 Both neural crest and nonneural crest-derived tissues express N-CAM protein and mRNA in our study. Langley et al 17'18 found expression of N-CAM protein in rat endocrine cells of both neural crest and non-neural crest-derived tissues, including a rat insulinoma cell line. Our results also illustrate the wide distribution of N-CAM in most normal and neoplastic endocrine tissues. Interestingly, some parathyroid adenomas
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Figure 3. In situ hybridization results with the N-CAM oligonucleotide probe. A:
Pheochromocytoma showing a positive hybridization signal (X300). B: In situ hybridization with the sense of oligonucleotide probeshowing no specifichybridization signal (x300).
had N-CAM mRNA but no detectable N-CAM protein, which probably reflects the greater sensitivity of the ISH analysis. The negative immunostaining of the parathyroid adenoma for N-CAM may reflect rapid turnover of the protein in these tumors. A parathyroid adenoma was also negative for N-CAM protein by immunoblot studies. In addition, a medullary thyroid carcinoma that had immunoreactive N-CAM by immunostaining was also negative by immunoblot analysis. Failure to detect N-CAM protein by immunoblotting may be related to the loss of activity of N-CAM in this tumor during extraction. Tumor heterogeneity in the expression of N-CAM protein was also observed in some tumors, such as the two pheochromocytomas analyzed by immunoblotting. The distribution of N-CAM protein and mRNA in many neuroendocrine cells and tumors suggest that N-CAM may be useful diagnostically. Although the antibodies
used in this study worked best in frozen sections, other workers developed N-CAM antibodies that recognize determinants that are preserved in formalin-fixed tissue sections.19 These investigators reported that N-CAM could be used to distinguish between small cell and nonsmall cell lung carcinoma.19 Patel et al32 recently reported that monoclonal antibody UJ13A recognized NCAM and that immunoreactivity was found in a variety of pediatric tumors, including Wilms' tumor, neuroblastoma, rhabdomyosarcoma, and Ewing's sarcoma.32 Broadspectrum neuroendocrine markers such as neuronspecific enolase,33 chromogranin A,3 and synaptophysin35 are related to specific characteristics of neuroendocrine cells, such as specific cytoplasmic enzymes or secretory products. N-CAM is a cell surface sialoglycoprotein that mediates homophilic binding between cells.36 Based on our studies and previous reports,17.18
N-CAM Expression in Normal and Neoplastic Human Neuroendocrine Tissues 967 AJP Ail 1.991, Vol. 138, No. 4
Figure 4. Neuroblastoma showing a positive hybridization signal with the N-CAM oligonucleotide probe (x300).
N-CAM may be another potential broad-spectrum marker for neuroendocrine cells and tumors. The variation in the amounts of N-CAM protein seen on Western blotting studies of the two pheochromocytomas probably reflect the heterogeneity common for many antigens in tumors. The localization of N-CAM antigen by IHC on cellsurface membrane was consistent with previous reports and ultrastructural observation.17-19 The limited extracellular staining on basement membrane or collagen fibrils have previously been reported.1819'37 It has been suggested that N-CAM determinants lost from the cell surface would adhere to neighboring components by a heparin-binding mechanism.18 In situ hybridization analysis provided further insight Figure 5. Pituitary null cell adenoma also
exressed N-CAM mRNA in some cells (X300).
into the expression of N-CAM in neuroendocrine tissues. The strongest ISH signals were found in pheochromocytomas, neuroblastomas, and paragangliomas, suggesting that more N-CAM mRNA might be expressed in tumors of neural crest origin compared with those of nonneural crest origin. Prieto et a138 localized N-CAM polypeptides and mRNA in various non-neural tissues during embryonic development, but the mRNA of the large cytoplasmic domain (Id) of N-CAM was found only in neural tissues. The various isoforms of N-CAM reflect its molecular complexity, as antibodies and molecular probes have been used to show that alternative splicing of a single N-CAM gene can generate up to four main classes of N-CAM polypeptides.6 The functional advantages of the diversity in the primary structure of the N-
968 Jin, Hemperly, and Lloyd AP APril 1991, Vol. 138, No. 4 16
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14
known, further studies on the regulation of N-CAM expression in normal cells and in various neoplastic tissues should provide new information about the role of N-CAM in normal and neoplastic tissues.
2
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References
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Figure 6. The distribution of silver grains per cell in normal and neoplastic tissues. The distribution of silver grains per cellfor 100 to 150 cells was counted and subtractedfrom the background to correct for nonspecific hybridization. The cases include 1) pheochromocytoma (n = 2); 2) neuroblastoma (n = 2); 3) pituitary null cell adenoma (n = 2); 4) medullary thyroid carcinoma (n = 2); 5) small cell lung carcinoma (n = 1); 6) gastrinoma (n = 1); 7) insulinoma (n = 1); 8) paratyroid adenoma (n = 2); 9) normalpituitary (n = 2); 10) normal adrenal (n = 2); and 11) normal liver (n = 1). The bars represent the SEM
CAM molecule is unknown. Recent studies by Doherty et
al,39 in which full-length cDNAs for a variety of human N-CAM isoforms were transfected into mouse L3 and NIH 3T3 cells, showed that both transneuronal and lipidlinked N-CAM isoforms were components of the regulator molecules that determined neuronal morphology and process outgrowth. The regulation of the different N-CAM glycoproteins in various tissues is not well known. N-CAM with a molecular weight of 180 kd and a low polysialic acid (PSA) content is characteristic of differentiated postmitotic neurons in adult neural system. N-CAM 140 kd with low PSA is more widely distributed in embryos and adults.10'11 Recently several studies have demonstrated that the high PSA forms of N-CAM (180 kd) could be expressed in some non-neural tumors or cell lines.1723 Roth et al22 reported that the high-PSA form of N-CAM (about 200 kd) was expressed in Wilms' tumors but not in normal adult kidney. In our studies, tumors analyzed by Western blots shared both 180-kd and 1 40-kd proteins with adult brain. It has been suggested that N-CAM with high PSA can inhibit cell-cell interactions and reduce cell adhesion, and could therefore lead to a more invasive growth of some neoplasms.203640 Several recent studies have illustrated the utility of N-CAM proteins as potential diagnostic tools for neuroendocrine neoplasms such as small cell lung carcinomas and pituitary adenomas.41'42 N-CAM plays an important role in cell proliferation, migration, and differentiation. Although the significance of the various forms of N-CAM in different tissues is un-
1. Edelman GM: Cell adhesion molecules in the regulation of animal form and tissue pattern. Ann Rev Cell Biol 1986, 2:81-116 2. Nybroe 0, Linnemann D, Bock E: N-CAM biosynthesis in brain. Neurochem Int 1988,12:251-262 3. Hemperly JJ, Murray BA, Edelman GM, Cunningham BA: Sequence of a cDNA clone encoding the polysialic acid rich and cytoplasmic domains of the neural cell adhesion molecule N-CAM. Proc Natl Acad Sci USA 1986, 83:3037-3041 4. Williams AF, Barclay AN: The immunoglobulin superfamily domains for cell surface recognization. Ann Rev Immunol 1988, 6:381-406 5. Owens GC, Edelman GM, Cunningham BA. Organization of the neural cell adhesion molecule (N-CAM) gene: Altemative exon usage as the basis for different membraneassociated domains. Proc Natl Acad Sci USA 1987, 84:294-298 6. Cunningham BA, Hemperly JJ, Murray BA, Prediger EA, Brackenbury R, Edelman GM: Neural cell adhesion molecule: Structure, immunoglobulin-like domains, cell surface modualtion, and alternative RNA splicing. Science 1987,
236:799-806 7. Jorgensen OS, Bock E: Brain specific synaptosomal membrane proteins demonstrated by crossed immunoelectro-
phoresis. J Neurochem 1974, 23:879-880 8. Goridis C, Hirn M, Langley OK, Ghandour S, Gombos G: Brain cell surface glycoproteins identified by monoclonal antibodies. Prog Brain Res 1983, 58:201-208 9. Noble M, Albrechtsen M, Moller C, Lyles J, Bock E, Goridis C, Watanabe M, Rutishauser U: Glial cells express NCAM/D2-CAM like polypeptides in vitro. Nature 1985, 316:725-728 10. Gennarini G, Hirsch MR, He HT, Him M, Finne, Goridis C: Differential expression of mouse neural cell adhesion molecule (N-CAM) mRNA species during brain development and in neural cell lines. J Neurosci 1986, 6:1983-1990 11. Murray BA, Owens GC, Prediger EA, Crossin KL, Cunningham BA, Edelman GM: Cell surface modulation of the neural cell adhesion molecule resulting from alternative mRNA splicing in a tissue-specific developmental sequence. J Cell Biol 1986,103:1431-1439 12. Crossin KL, Chuong CM, Edelman GM: Expression sequences of cell adhesion molecule. Proc NatI Acad Sci USA 1985, 82:6942-6946 13. Akeson RA, Wujek JR, Roe S, Warren SL, Small SJ: Smooth muscle cells transiently express NCAM. Mol Brain Res 1988, 4:107-120 14. Thor G, Probstmeier R, Schachner M: Characterization of the cell adhesion molecules Li, N-CAM and Jl in the mouse intestine. EMBO J 1987, 6:2581-2586
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15. Thomas PS, Pietranjeli CE, Hayashi SI, Schachner M, Goridis C, Low M, Kincade PW: Demonstration of neural cell adhesion molecules on stromal cells that support lymphopoiesis. Leukemia 1988; 2:171-175 16. Lanier LL, Testi R, Bindl J, Phillips JH: Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule. J Exp Med 1989, 169:2233-2238 17. Langley OK, Aletsee MC, Gratzl M: Endocrine cells share expression of N-CAM with neurones. FEBS Lett 1987, 220:108-112 18. Langley OK, Aletsee-Ufrecht MC, Grant NJ, Gratzl M: Expression of the neural cell adhesion molecule NCAM in endocrine cells. J Histochem Cytochem 1989, 37:781-791 19. Kibbelaar RE, Moolenaar CEC, Michalides RJAM, BitterSuermann D, Addis B, Mooi WJ: Expression of the embryonal neural cell adhesion molecule N-CAM in lung carcinoma. Diagnostic usefulness of monoclonal antibody 735 for the distribution between small cell and non-small cell lung carcinoma. J Pathol 1989; 159:23-28 20. Kitty Moolenaar CEC, Muller EJ, Schol DJ, Figdor CG, Bock E, Bitter-Snermann D, Michalides RJAM: Expression of neural cell adhesion molecule-related sialoglycoprotein in small cell lung cancer and neuroblastoma cell lines H69 and CHP-212. Cancer Res 1990; 50:1102-1106 21. Patel K, Moore SE, Dickson G, Rossell RJ, Beverley PC, Kemshead JT, Walsh FS: Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small cell lung carcinoma and neuroblastoma. Int J Cancer 1989, 44:573-578 22. Roth J, Zuber C, Wagner P, Taatjes DJ, Weisgerber C, Heitz PU, Goridis C, Bitter-Suermann D: Reexpression of poly (sialic acid) units of the neural cell adhesion molecule in Wilms Tumor. Proc Natl Acad Sci USA 1988,85:2999-3003 23. Lipinski M, Hirsch MR, Deagostini-Bazin H, Yamada 0, Tursz T, Goridis C: Characterization of neural cell adhesion molecule (NCAM) expressed by Ewing and neuroblastoma cell lines. Int J Cancer 1987;,40:81-86 24. Cole SPC, Mirski S, McGarry RC, Cheng R, Campling BG, Rode JC: Differential expression of the Leu-7 antigen on human lung tumor cells. Cancer Res 1985, 45:4285-4290 25. Kunemund V, Jungalwala FB, Fischer G, Chou DKH, Keilhauer G, Schachner M: The L2/HNK-1 carbohydrate of neural cell adhesion molecules is involved in cell interactions. J Cell Biol 1988,106:213-223 26. Hemperly JJ, DeGuglielmo JK, Reid RA: Characterization of cDNA clones defining variant forms of the human neural cell adhesion molecule N-CAM. J Mol Neuroscience (In press) 27. Lloyd RV, Coleman K, Fields K, Nath V: Analysis of prolactin and growth hormone production in hyperplastic and neoplastic rat pituitary tissues by the hemolytic plaque assay. Cancer Res 1987, 47:1087-1092 28. Lloyd RV, lacangelo A, Eiden LE, Cano M, Jin L, Grimes M: Chromogranin A and B messenger ribonucleic acids in pi-
tuitary and other normal and neoplastic human endocrine tissues. Lab Invest 1989, 60:548-556 29. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685 30. Towbin H. Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedures and some applications. Proc Natl Acad Sci USA 1979, 76:4350-4354 31. LeDouarin NM: The Neural Crest. Cambridge, UK, Cambridge University Press, 1982 32. Patel K, Rossell RJ, Bourne S, Moore SE, Walsh FS, Kemshead JT: Monoclonal antibody UJ13A recognizes the neural cell adhesion molecule (N-CAM). Int J Cancer 1989, 44:1062-1068 33. Schmechel D, Marangos PJ, Brightman M: Neuron-specific enolase in a molecular marker for peripheral and central neuroendocrine cells. Nature 1978, 276:834-836 34. Lloyd RV, Wilson BS: Specific endocrine tissue marker defined by a monoclonal antibody. Science 1983, 222:628630 35. Navone F, Jahn R, Di Gioia G, Stukenbrok H, Greengard P, De Camilli P: Protein 38: An integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J Cell Biol 1986,103:2511-2527 36. Rutishauser U, Acheson A, Hall AK, Mann DM, Sunshine J: The neural cell adhesion molecule (NCAM) as a regulator of cell-cell interactions. Science 1988, 240:53-57 37. Sanes JR, Schachner M, Covault J: Expression of several adhesion macromolecules (N-CAM, Ll, J1, NILE, uvomorulin, laminin, fibronectin, and a heparan sulfate proteoglycan) in embryonic, adult, and denervated adult skeletal muscle. J Cell Biol 1986, 102:420-431 38. Prieto AL, Crossin KL, Cunningham BA, Edelman GM: Localization of mRNA for neural cell adhesion molecule (NCAM) polypeptides in neural and non-neural tissues by in situ hybridization. Proc Natl Acad Sci USA 1989, 86:95799583 39. Doherty P, Barton CH, Dickson G, Seaton P, Rowett LH, Moore SG, Gower HJ, Walsh FS: Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms. J Cell Biol 1989,109:789-798 40. Hoffman S, Edelman GM: Kinetics of homophilic binding of embryonic and adult forms of the neural cell adhesion molecule. Proc Natl Scand Sci USA 1983, 80:5762-5766 41. Aletsee-Ufrecht MC, Langley K, Rotsch M, Havemann K, Gratzl M: NCAM: a surface marker for human small cell lung cancer cells. FEBS Lett. 1990, 267:295-300 42. Aletsee-Ufrecht MC, Langley K, Gratzl 0, Gratzl M: Differential expression of the neural cell adhesion molecule NCAM 140 in human pituitary tumors. FEBS Left 1990,
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Expression of Neural Cell Adhesion Molecule in Normal and Neoplastic Human Neuroendocrine Tissues Long Jin,* John J. Hemperly,t and Ricardo V. Lloyd* From the Department of Pathology, * University of Michigan, Ann Arbor, Michigan; and Becton Dickinson and Company, t Research Triangle Park, North Carolina
The neural cell adhesion molecule (N-CAM) is a group of cell surface glycoproteins involved in direct cell-cell adhesion. N-CAM expression in normal and neoplastic tissues was examined with specific antibodies and oligonucleotide probes by immunohistochemistry and in situ hybridization. Most neuroendocrine cells and tumors with secretory granules expressed N-CAM protein and mRNA Parathyroid adenomas (4) were somewhat unusua* because N-CAM mRNA, but not proteing was detected in some of these benign neoplasms. Most non-neuroendocrine cells and tumors did not express N-CAM, although uterine smooth muscle and an adrenal cortical carcinoma were both positive. Western blots disclosed proteins of 180, 140, and 120 kd in normal adult brain; whereas two pheochromocytomas, a null cell adenoma and a gastrinoma had proteins of approximately 180 and 140 kd. These results indicate that N-CAM protein and mRIVA are widely expressed in neuroendocrine cells and neoplasms. N-CAM oligonucleotide probes as well as antibodies against N-CAM can be used as broad-spectrum neuroendocrine markers. In addition; these molecular probes can be used to examine the role of N-CAM in the development and regulation of neuroendocrine tissues. (Am J Pathol 1991, 138:961-969)
The neural cell adhesion molecule (N-CAM) is a group of cell surface glycoproteins involved in direct cel-cell adhesion.1" Cloning and sequencing of the cDNA for N-CAM indicates that N-CAM is a member of the immunoglobulin gene superfamily.4 Various forms of N-CAM have been found in vertebrate species such as chickens, mice, rats, and humans. In adult brain, N-CAM consists of three major polypeptides with Mr 180, 140, and 120 kd,
which are generated by alterative RNA splicing from a single gene.5'6 The sequence of the coding regions of the N-CAM gene has been well conserved during
evolution.1' 3 N-CAM originally was described as a D2 protein and brain-specific antigen-2 (BSP-2).7 It was later demonstrated that N-CAM, D2, and BSP-2 were immunochemically identical.9 The expression of N-CAM is regulated in a developmental stage and tissue-type-specific
manner.1012 In early embryos, the cells of all three primitive layers express N-CAM, whereas in adults N-CAM expression is more restricted. The expression of N-CAM in adult tissues was previously considered to be mainly restricted to the nervous system. Recent studies have shown, however, that N-CAM was also expressed in some non-neural tissues,1-16 including some endocrine cells.17'18 A few reports have described the presence of N-CAM in selected normal and pathologic human tissues.1'23 Moolenaar et al19'20 reported that an antiN-CAM monoclonal antibody 735 and small cell lung carcinoma (SCLC) cluster 1 monoclonal antibodies recognized identical glycoproteins in SCLC. The Leu-7-antigen on lung tumor cells, which is recognized by HNK-1 monoclonal antibody, is a carbohydrate epitope found on N-CAM, as well as on a number of other cell adhesion molecules.24'25 Recently polyclonal antisera against human N-CAM were prepared by one of us (John J. Hemperly). Using affinity-purified antisera, we have analyzed the distribution of N-CAM by immunohistochemistry (IHC) and immunoblot methods in neuroendocrine tissues. In addition, the DNA sequence of human N-CAM has been determined by the characterization of cDNA clones encoding N-CAM from a human neuroblastoma cDNA library.26 Various oligonucleotide probes to N-CAM were developed, and the distribution of N-CAM mRNA was analyzed by in situ hybridization (ISH). In this study, we have analyzed the distribution of N-CAM expression in Supported in part by NIH grant CA 37238. Accepted for publication December 19, 1990. Address correspondence to R. Lloyd, MD, PhD, Department of Pathology, University of Michigan Medical Center, 1500 E. Medical Center Dr., Room 2G332/Box 0054, Ann Arbor, Ml 48109-0054.
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normal and neoplastic human tissues at both protein and mRNA levels. The results showed a wide distribution of N-CAM expression in human neuroendocrine tissues.
Materials and Methods Tissues Fifty-five normal and neoplastic human tissues from mostly surgical specimens and a few autopsy specimens from the Department of Pathology were used in these studies (Table 1). Tissues were frozen in liquid nitrogen and kept at - 700C for up to 2 years. For IHC and ISH studies, the tissues were embedded in optimal cutting temperature (OCT) medium and approximately 6- to 8- -thick sections were cut in a cryostat at - 200C. Sections used for IHC were mounted on poly-l-lysine-coated slides and fixed in acetone at room temperature for 10 minutes, and then kept in phosphate-buffered saline (PBS) until immunostaining was carried out. The sections used for ISH were mounted on 3-aminopropylmethoxysilane-coated slides, followed by fixation in 4% paraformaldehyde, pH 7.2, for 20 minutes. The slides were then washed in 2x SSC (1 x SSC = 0.15 mol/l [molar] NaCI and 0.015 mol/l Na citrate), dehydrated in 95% and 100% ethanol, and stored at - 700C for up to 1 month.
Immunohistochemistry The frozen sections were immunostained as previously reported using the ABC method27 (Vector Kits, Burlingame, CA). An immunoaffinity-purified rabbit polyclonal anti-human N-CAM antibody was used at 1/50 1/100 on all slides. The immunoaffinity-purified anti-N-CAM antibodies were prepared as follows. Human brain N-CAM was purified by immunoaffinity chromatography of a detergent extract on a column of monoclonal antibody antiLeu19 (Becton Dickinson) coupled to Sepharose. AntiLeul 9 reacts with N-CAM as well as a subset of cytotoxic T cells in the peripheral blood. Bound material was eluted with 0.1 mol/l diethanolamine (pH 11), 0.5% NP-40, and immediately neutralized with sodium phosphate. Purified N-CAM was coupled to Reacti-Gel 6X (Pierce Chemical Co.) as recommended by the manufacturer. Briefly, the relatively dilute N-CAM eluate from the anti-Leu19Sepharose was dialyzed against 0.1 molA sodium borate (pH 8.5) and mixed with the beads. After absorbing overnight, the beads were washed and stored. To purify the antibodies, an antisera prepared by immunizing rabbits with N-CAM purified as above was rocked with about 1 ml of N-CAM-Reacti-Gel for 1 hour in the cold. The column was washed with PBS. The antibodies were eluted with 3 ml of pH 11 buffer without detergent and neutralized with 2 mol/l sodium phosphate. The protein concen-
Table 1. N-CAM Immunobistochemical and In Situ Hybridization Analys* of Nornmal and Neoplastic Human Tissues In situ Tissues Diagnosis Immunohistochemistry hybridization
Pituitary Adrenal
Cerebral cortex Cerebellum Thyroid
Parathyroid Pancreas
Normal Null cell Adenoma Prolactin adenoma Normal Pheochromocytoma Cortical Carcinoma Neuroblastoma Paraganglion/paraganglioma Normal Normal Medullary carcinoma Adenocarcinoma Adenoma Normal Insulinoma/gastrinoma
Stomach Lung
Carcinoid Carcinoid Small cell carcinoma
Skin Breast Ovaries Uterus
Merkel cell carcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenomyosis (myometrium) Adenocarcinoma Normal Normal Squamous cell carcinoma
Colon Liver Skin
* Number of positive cases/number of total cases examined. t Represent focal positive staining in less than 25% of the tumor cells.
3/3* 4/4 1/1 3/3 4/4 1/1 2/2 3/3 1/1 2/2 3/3 0/2 0/4 2/2 2/2 1/1 1/1 1/1 1/1 3/4t 0/3 0/1 1/1 0/2 0/1 0/1 0/1
3/3* 4/4 1/1 3/3 4/4 1/1 2/2 3/3 1/1 2/2 3/3 0/2 2/4' 1/2 2/2 0/1 1/1 1/1 1/1 3/4t 0/3 0/1 1/1 0/2 0/1 0/1 0/1
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tration of the antibody was about 80 ,ug/ml. Normal rabbit serum diluted 1/100 was substituted for primary antibody as a negative control.
Probes and Labeling An oligonucleotide probe that recognizes messenger RNA (mRNA) coding for N-CAM was prepared on the basis of human N-CAM cDNA sequence. This 30-base oligonucleotide probe was complementary to human NCAM, encoding for amino acids 808 through 817 of the cytoplasmic domain: 5' d(GGC TTC GTT TCT GTC TCC TGG CAC TCT GGC)-3'.26 This sequence was present on N-CAM 180 and 140. The probe was prepared using an Applied Biosystems (Foster City, CA) 381 A DNA synthesizer and purified using an OPC cartridge (Applied Biosystems). The oligonucleotide probe was labeled by the 3'-end labeling method as previously described,28 using 100 ng of oligonucleotide incubated with 45 ,uCi of [35S] dCTP or [35S] dATP (1300 1500 ci/mmol), 2 mmol/I (millimolar) CaCI2, 0.5 mmol/I dithiothreitol, and 20 units of terminal deoxynucleotidyl transferase in 100 mmol/l potassium cacodylate (pH 7.2) for 45 minutes at 370C. The labeled probe was purified by gel electrophoresis on 12% polyacrylamide gels as previously described.28.
and 100% ethanol with 0.3 mol/l ammonium acetate, and air dried. Autoradiography was carried out by dipping in Kodak NTB2 emulsion diluted 1:1 with distilled water containing 0.3 moVI ammonium acetate. The slides were allowed to air dry for 1 hour and then placed in a 40C dessicated chamber for 1 week. They were subsequently developed for 3 minutes in Kodak D-19 developer, washed in water for 1 minute and fixed for 5 minutes in Kodak Fixer. After washing in water for 1 hour, they were stained with hematoxylin and eosin (H&E), and coverslipped. The results of ISH were analyzed under the microscope by using dark-field and bright-field illumination. The control for ISH consisted of 1) prior digestion of frozen sections with 100 ,ug/ml of RNAse A (Sigma) at 370C for 45 minutes before ISH; 2) omission of specific probes in hybridization buffer for ISH; 3) Using a sense DNA oligonucleotide N-CAM probe. Quantitation of ISH data was done by taking photographs at a final magnification of x 1000. Grains per cell were enumerated using a total of 100 to 150 cells. Results were expressed as number of silver grains per cell.
-
In Situ Hybridization Slides were incubated in 100% and 95% ethanol for 5 minutes each and washed in water at room temperature. The slides were then treated with 0.25% acetic anhydride in triethanolamine (vol/vol) for 10 minutes followed by incubation in 2x SSC for 30 minutes at 700C, then washed in 2 x SSC at room temperature for 5 minutes before covering the slides with 40 ,ul of hybridization buffer (2x SSC containing Denhardt's solution [0.02% Ficoll/0.02% bovine serum albumin/0.02% polyvinylpryrrolidine], 10% dextran sulfate, 125 ,ug/ml of yeast tRNA, 100 RIl/ml of sonicated salmon sperm DNA, and 50% formamide in 20 mmol/I sodium phosphate [pH 7.2]) for 1 hour at room temperature. The labeled probes were then diluted in hybridization buffer containing 20 mmol/I dithiothreitol and 1 x 1 o6 cpm of probe/slide. They were covered with coverslips treated with Sigmacote and incubated overnight at 440C in moist chambers. After 18 hours, the coverslips were gently removed in 2x SSC and the slides were washed at room temperature in 2x SSC for 2 hours, then in 1 x SSC for 1 hour and 0.5x SSC for 30 minutes. A subsequent wash in 0.5x SSC for 30 minutes at 440C was followed by a 30-minute wash at room temperature in 0.5x SSC. The slides were then dehydrated in 95%
Immunoblot Proteins were extracted with 1% NP40 or 1% sodium dodecyl sulfate, and separated on a polyacrylamide gel, transferred to nitrocellulose paper, and immunostained with a polyclonal N-CAM antibody."9 30 Proteins were not treated with neuraminidase before Western blotting.
Results
Immunohistochemistry Most normal and neoplastic neuroendocrine tissues showed diffuse or focal immunoreactivity with the polyclonal N-CAM antiserum, including pituitary adenomas, pheochromocytomas, neuroblastomas, medullary thyroid carcinomas, and other endocrine tumors from lung, pancreas, gastrointestinal tract, and skin (Table 1) (Figures 1 A-F). Immunoreactivity appeared to be associated with the cytoplasmic membranes, in some cases with better morphologic preservation of tissues (Figures 1 C, D). The normal adrenal medullae and pheochromocytomas were strongly labeled, but adrenal cortical cells were negative (Figure 1A, B). One adrenal cortical carcinoma examined, however, was strongly positive. Normal pituitary (some secretory cells of adenohypophysis and the neurohypophysis) (Figure 1 C), brain (the neuron, astrocytes, and fiber bundles), and cerebellum (the Purkinje cells and granular layer) from autopsy were also intensely stained. All cells of the pancreatic islets of Langerhans
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Figure 1. A: (top left) Immunoristocbemknal staining of an acetone-fixed frozen section of adrenal gland showing a positive reaction for N-CAM protein in the medulla while the cortex is negative (x250). B: (top right) Pheochromocytoma localizing N-CAM tumor cells (X400). C: (middle left) Localization of N-CAM in the adenoypopbysis of normal pituitary tissues. Many of the cells stained positively (X350). D: (middle right) N-CAM immunoreactivty in a medullary thyroid carcinoma (x350). E: (bottom left) Merkel cell carcinoma sbowing positive staining for N-CAM in mosttumor cells (x300). F: (bottom right) Parathyroid adenomas did not sbow positive immunoreactivity with the
N-CAM antibody (x300).
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were stained. Medullary thyroid carcinomas (Figure 1 D) and a Merkel cell carcinoma (Figure 1 E) were positive. All four cases of the parathyroid adenomas were completely negative after immunostaining (Figure 1 F). In contrast, most nonendocrine tissues, such as liver, skin, adult muscle, colon, and ovarian carcinomas did not react with the N-CAM antiserum. Smooth muscle from a case of uterine adenomyosis stained positively, whereas the blood vessel smooth muscle in the same case was negative. Several cases of breast carcinomas had focal immunostaining of the epithelial cells. Extracellular staining was also seen on some elements of the basement membrane, collagen fibrils, and some lymphocytes, as well as nerve fibers.
Immunoblot N-CAM determinants were analyzed in different tissues by immunoblot using the polyclonal N-CAM antibody (Figure 2). Pituitary null cell adenomas, gastrinomas, and two pheochromocytomas were positive with two major bands of approximately 180 kd and 140 kd. Adult brain, used as a positive control, had three positive bands of 180, 140, and 120 kd. Liver tissue, a parathyroid adenoma, and a medullary thyroid carcinoma were negative by immunoblot studies. The extra bands present in the Western blots in lanes 1 and 6 and in the two pheochro-
mocytomas (lanes 3 and 4) were detected consistently with the polyclonal antiserum and with a battery of monoclonal antibodies to N-CAM. These bands probably represent variation in molecular sizes due to differential glycosylation.
In Situ Hybridization Analysis of the distribution of N-CAM mRNA by ISH with the N-CAM oligonucleotide probe is summarized in Table 1 (Figures 3 to 5). The results of ISH demonstrated that most endocrine tissues expressed N-CAM mRNA, which correlated with the IHC analysis. Silver grains were present over the nucleus and the cytoplasm (Figures 3 through 5). Pheochromocytomas, neuroblastomas, and pituitary null cell adenomas had stronger hybridization signals than other endocrine tissues, as judged by the number of silver grains per cell (Figure 6). The normal adrenals and pituitaries had weaker hybridization signals compared with the tumors that originated from these tissues. Positive hybridization signals were found focally in two of four parathyroid adenomas. No ISH signals were detected in most non-neuroendocrine tissues. Hybridization with the sense N-CAM probe did not produce a positive hybridization signal (Figure 3B). Omission of the oligonucleotide probe or pretreatment with RNAse before ISH abolished the hybridization signal.
Discussion
211 -
107-
Figure 2. Immunoblot showing the expression of various N-CAM species in endocrine tumors and adult brain tissues. Lane 1 is a pituitary null cell adenoma: lane 2 is a gastrinoma; lanes 3 and 4 are pheochromocytomas; lane 5 shows normal liver and lane 6 shows normal adult brain. Three hundred micrograms ofprotein was used in lanes 1 to 5, and 100 jig ofprotein was used in lane 6 The molecular weight standards are shown on the left.
The present studies demonstrate that N-CAM polypeptides and mRNA are present in many neuroendocrine cells and tumors. Early studies suggested that N-CAM expression was restricted to cells of neural crest lineage, such as neurons, glial cells, chromaffin cells of the adrenal medullary, and some tumor cell lines derived from these.2'710 Because neuroendocrine cells have many features in common with neural cells such as the production of various hormones and amines, we extended the study of N-CAM distribution to many neuroendocrine tissues. The adrenal medulla, paraganglia, neurohypophysis, and C cells of the thyroid are neuroendocrine tissues with a neural crest origin, whereas the neuroendocrine cells from pancreas, gastrointestinal tract, and lung are not of neural crest origin.31 Both neural crest and nonneural crest-derived tissues express N-CAM protein and mRNA in our study. Langley et al 17'18 found expression of N-CAM protein in rat endocrine cells of both neural crest and non-neural crest-derived tissues, including a rat insulinoma cell line. Our results also illustrate the wide distribution of N-CAM in most normal and neoplastic endocrine tissues. Interestingly, some parathyroid adenomas
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Jin, Hemperly, and Lloyd
AJP Apil 1991, Vol. 138, No. 4
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Figure 3. In situ hybridization results with the N-CAM oligonucleotide probe. A:
Pheochromocytoma showing a positive hybridization signal (X300). B: In situ hybridization with the sense of oligonucleotide probeshowing no specifichybridization signal (x300).
had N-CAM mRNA but no detectable N-CAM protein, which probably reflects the greater sensitivity of the ISH analysis. The negative immunostaining of the parathyroid adenoma for N-CAM may reflect rapid turnover of the protein in these tumors. A parathyroid adenoma was also negative for N-CAM protein by immunoblot studies. In addition, a medullary thyroid carcinoma that had immunoreactive N-CAM by immunostaining was also negative by immunoblot analysis. Failure to detect N-CAM protein by immunoblotting may be related to the loss of activity of N-CAM in this tumor during extraction. Tumor heterogeneity in the expression of N-CAM protein was also observed in some tumors, such as the two pheochromocytomas analyzed by immunoblotting. The distribution of N-CAM protein and mRNA in many neuroendocrine cells and tumors suggest that N-CAM may be useful diagnostically. Although the antibodies
used in this study worked best in frozen sections, other workers developed N-CAM antibodies that recognize determinants that are preserved in formalin-fixed tissue sections.19 These investigators reported that N-CAM could be used to distinguish between small cell and nonsmall cell lung carcinoma.19 Patel et al32 recently reported that monoclonal antibody UJ13A recognized NCAM and that immunoreactivity was found in a variety of pediatric tumors, including Wilms' tumor, neuroblastoma, rhabdomyosarcoma, and Ewing's sarcoma.32 Broadspectrum neuroendocrine markers such as neuronspecific enolase,33 chromogranin A,3 and synaptophysin35 are related to specific characteristics of neuroendocrine cells, such as specific cytoplasmic enzymes or secretory products. N-CAM is a cell surface sialoglycoprotein that mediates homophilic binding between cells.36 Based on our studies and previous reports,17.18
N-CAM Expression in Normal and Neoplastic Human Neuroendocrine Tissues 967 AJP Ail 1.991, Vol. 138, No. 4
Figure 4. Neuroblastoma showing a positive hybridization signal with the N-CAM oligonucleotide probe (x300).
N-CAM may be another potential broad-spectrum marker for neuroendocrine cells and tumors. The variation in the amounts of N-CAM protein seen on Western blotting studies of the two pheochromocytomas probably reflect the heterogeneity common for many antigens in tumors. The localization of N-CAM antigen by IHC on cellsurface membrane was consistent with previous reports and ultrastructural observation.17-19 The limited extracellular staining on basement membrane or collagen fibrils have previously been reported.1819'37 It has been suggested that N-CAM determinants lost from the cell surface would adhere to neighboring components by a heparin-binding mechanism.18 In situ hybridization analysis provided further insight Figure 5. Pituitary null cell adenoma also
exressed N-CAM mRNA in some cells (X300).
into the expression of N-CAM in neuroendocrine tissues. The strongest ISH signals were found in pheochromocytomas, neuroblastomas, and paragangliomas, suggesting that more N-CAM mRNA might be expressed in tumors of neural crest origin compared with those of nonneural crest origin. Prieto et a138 localized N-CAM polypeptides and mRNA in various non-neural tissues during embryonic development, but the mRNA of the large cytoplasmic domain (Id) of N-CAM was found only in neural tissues. The various isoforms of N-CAM reflect its molecular complexity, as antibodies and molecular probes have been used to show that alternative splicing of a single N-CAM gene can generate up to four main classes of N-CAM polypeptides.6 The functional advantages of the diversity in the primary structure of the N-
968 Jin, Hemperly, and Lloyd AP APril 1991, Vol. 138, No. 4 16
T
14
known, further studies on the regulation of N-CAM expression in normal cells and in various neoplastic tissues should provide new information about the role of N-CAM in normal and neoplastic tissues.
2
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References
8
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1
2
3
4
5
6
7
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10
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Figure 6. The distribution of silver grains per cell in normal and neoplastic tissues. The distribution of silver grains per cellfor 100 to 150 cells was counted and subtractedfrom the background to correct for nonspecific hybridization. The cases include 1) pheochromocytoma (n = 2); 2) neuroblastoma (n = 2); 3) pituitary null cell adenoma (n = 2); 4) medullary thyroid carcinoma (n = 2); 5) small cell lung carcinoma (n = 1); 6) gastrinoma (n = 1); 7) insulinoma (n = 1); 8) paratyroid adenoma (n = 2); 9) normalpituitary (n = 2); 10) normal adrenal (n = 2); and 11) normal liver (n = 1). The bars represent the SEM
CAM molecule is unknown. Recent studies by Doherty et
al,39 in which full-length cDNAs for a variety of human N-CAM isoforms were transfected into mouse L3 and NIH 3T3 cells, showed that both transneuronal and lipidlinked N-CAM isoforms were components of the regulator molecules that determined neuronal morphology and process outgrowth. The regulation of the different N-CAM glycoproteins in various tissues is not well known. N-CAM with a molecular weight of 180 kd and a low polysialic acid (PSA) content is characteristic of differentiated postmitotic neurons in adult neural system. N-CAM 140 kd with low PSA is more widely distributed in embryos and adults.10'11 Recently several studies have demonstrated that the high PSA forms of N-CAM (180 kd) could be expressed in some non-neural tumors or cell lines.1723 Roth et al22 reported that the high-PSA form of N-CAM (about 200 kd) was expressed in Wilms' tumors but not in normal adult kidney. In our studies, tumors analyzed by Western blots shared both 180-kd and 1 40-kd proteins with adult brain. It has been suggested that N-CAM with high PSA can inhibit cell-cell interactions and reduce cell adhesion, and could therefore lead to a more invasive growth of some neoplasms.203640 Several recent studies have illustrated the utility of N-CAM proteins as potential diagnostic tools for neuroendocrine neoplasms such as small cell lung carcinomas and pituitary adenomas.41'42 N-CAM plays an important role in cell proliferation, migration, and differentiation. Although the significance of the various forms of N-CAM in different tissues is un-
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